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J. Biol. Chem., Vol. 276, Issue 33, 31004-31015, August 17, 2001
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From the
Received for publication, January 2, 2001, and in revised form, June 25, 2001
The mammalian 70K protein, a component of
the U1 small nuclear ribonucleoprotein involved in pre-mRNA
splicing, interacts with a number of proteins important for regulating
constitutive and alternative splicing. Similar proteins that interact
with the yeast homolog of the 70K protein, Snp1p, have yet to be
identified. We used the two-hybrid system to find four
U1-Snp1 associating (Usa) proteins.
Two of these proteins physically associate with Snp1p as assayed by
coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal
component. This interaction suggests some novel functions for Snp1p and
the U1 small nuclear ribonucleoprotein late in spliceosome development.
The other, Exo84p, is a conserved subunit of the exocyst, an
eight-protein complex functioning in secretion. We show here that
Exo84p is also involved in pre-mRNA splicing. A
temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also
led to a defect in splicing and prespliceosome formation in
vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.
The U1 snRNP1 has an
early, hierarchic role in pre-mRNA splicing in the yeast
Saccharomyces cerevisiae (1-3). It must be bound to
pre-mRNA for subsequent stable association of the other four spliceosomal snRNPs with the pre-mRNA. Once U1 snRNP is bound, U2
snRNP binds, and the prespliceosome is formed. The tri-snRNP complex,
U4/U6.U5, then binds to form the spliceosome. The spliceosome next
undergoes a number of coordinated rearrangements (4). The duplexes
between the U4 and U6 snRNAs and between U1 snRNA and the 5'-splice
site (SS) of the pre-mRNA are disrupted, whereas new pairings
between U2 and U6 snRNAs, U6 snRNA and the 5'-SS, and U5 snRNA and the
pre-mRNA exons 1 and 2 are formed. These rearrangements lead to the
formation of the active catalytic site of the spliceosome. Splicing of
the pre-mRNA then ensues by two transesterification reactions.
The yeast U1 snRNP recognizes both the 5'-SS and the branchpoint region
of the pre-mRNA (3, 5). From 5 to 7 nucleotides of the 5' end of
the U1 snRNA base pair with the 5'-SS of the pre-mRNA to form the
short U1/5'-SS duplex (3). Additionally eight proteins of the U1 snRNP,
including the C and Snp1p proteins, contact the 5'-SS region and exon 1 and may stabilize the U1/5'-SS duplex (6). Proteins bound to the
pre-mRNAs branchpoint region associate with at least one protein
component of the yeast U1 snRNP, Prp40p, to form a bridge to the 5'-SS
(7). Similar protein-protein interactions suggest such a protein bridge
in metazoans as well (8).
During spliceosome formation, the 5'-SS switches its pairing from the
U1 to the U6 snRNA (4). It is not understood when and how the U1 snRNP
is displaced from the 5'-SS. It was first suggested that the U1 snRNP
physically dissociates from the developing spliceosome before the
tri-snRNP binds, but it is now thought that the displacement occurs
when the tri-snRNP enters the spliceosome or shortly thereafter
(9-11). One of the DEAD box helicases, Prp28p, may eliminate the
U1/5'-SS pairing by unwinding the duplex, by promoting the pairing of
U6 with the 5'-SS to displace U1, or by altering C protein binding to
destabilize the U1/5'-SS duplex (11, 12).
In metazoans, the U1 snRNP also functions in regulating both
constitutive and alternative splicing. An element of the U1 snRNP important in this function is the 70K protein that interacts with a
number of factors. The 70K protein binds to stem-loop 1 of the U1 snRNA
via an RNA recognition motif in its central domain. Its N-terminal
domain interacts with the U1 C protein to help stabilize the U1/5'-SS
duplex (13). Its C-terminal domain associates with the SR protein,
ASF/SF2 (14), and additional SR proteins (8, 15, 16). The SR proteins,
so-called because they contain serine-arginine dipeptide repeats,
enhance or repress splicing by recruiting or inhibiting other splicing
factors, especially the U1 and U2 snRNPs (17, 18). Some SR proteins
along with 70K form the protein bridge between the 5'-SS and the
branchpoint region (8). Several interactions between the SR proteins
and 70K are regulated by phosphorylation (14, 19), and several steps in
the splicing pathway, including spliceosome assembly, depend on the
phosphorylated state of these proteins (20). Both the SR (17, 21) and
70K (22) proteins are targets of specific kinases.
We considered that Snp1p (23), the yeast homolog of the 70K protein,
might also physically associate with proteins that regulate splicing in
yeast. Like the 70K protein with which it shares 30% identity, Snp1p
has three domains (23, 24), with the central domain binding via an RNA
recognition motif to the U1 snRNA (25). Snp1p is not required for
viability in all yeast strains (23, 24), but it is required for
efficient splicing (24), a phenotype suggestive of a regulatory role.
Unlike metazoans that have large numbers of SR proteins, yeast may have
only a few such proteins.
In this study we used the yeast two-hybrid system (26) to screen yeast
sequences for those encoding U1 Snp1p
associating (Usa) proteins. We found four Usa proteins. One
of these is Prp8p, a known, integral component of the U5 snRNP (27).
Another, Usa3p, has also been identified as Exo84p, a component of the
evolutionarily conserved exocyst complex of the secretory pathway (28).
Our characterization of a Ts exo84 mutant indicates that
Exo84p in vivo affects the expression of some genes with
introns and in vitro has a direct role in splicing. Both
interactions, Snp1p-Prp8p and Snp1p-Exo84p, may be involved in splicing
regulation. The Snp1p-Prp8p interaction may be important in
coordinating the 5'-SS switch from U1 to U6. The Snp1p-Exo84p
interaction may be involved in comodulating secretion and pre-mRNA
splicing. Intriguingly, Exo84p has two copies of the consensus target
site for an SR protein kinase.
The following enzymes, chemicals, and antibodies were obtained
from commercial suppliers as follows: restriction enzymes, Deep Vent
DNA Polymerase, T7 RNA polymerase, and T4 DNA ligase were from New
England Biolabs; avian myeloblastosis virus reverse transcriptase was from Life Sciences Research, Inc.; actinomycin D and
3-AT were from Sigma; RNasin RNase inhibitor, RQ1 RNase-free DNase, and
rabbit reticulocyte lysate were from Promega; zymolase was from
Seikagaku Corp.; Sequenase version 2.0 kit was from U. S. Biochemical
Corp.; DC protein assay kit was from Bio-Rad; anti-FLAG M2 immobilized
antibody was from Eastman Kodak Co.; [35S]methionine
(1000 Ci/mmol), [32P]UTP (3000 Ci/mmol), anti-mouse and
anti-rabbit IgG antibodies conjugated with peroxidase, chemiluminescent
detectors (ECL and ECL-plus), and Amplify fluorographic solution were
from Amersham Pharmacia Biotech; and anti-HA monoclonal antibody 16B12
and immobilized 16B12 antibody were from Babco, Inc. Anti-HA monoclonal
antibody 12CA5, originally from Babco, Inc., was a gift from V. Lundblad. Anti-Gal4p and anti-Bcy1p polyclonal antibodies were gifts
from J. Hopper and M. Werner-Washburne, respectively.
Media and Cell Manipulations
Standard yeast genetic methods (29) and media (30) were as
described. Plasmids were introduced by either transformation with PEG
(31) or by electroporation (32). When plasmids were introduced by
targeted integration (33), the site of integration was subsequently
confirmed by Southern analysis. For selecting bacterial isolates with a
LEU2-marked plasmid, we used M9 minimal medium supplemented
with 1 mM thiamine HCl, 100 mg/ml thymidine, 100 mg/ml
ampicillin, and the yeast amino acid mix lacking leucine (30).
Oligodeoxynucleotides
The oligos were synthesized by Genosys, Inc., or the University
of New Mexico Center for Genetics in Medicine. Their sequences are
available upon request.
Plasmids
The following plasmids were obtained from others as follows:
SNP1 on plasmids pRS3D and pDi (23) from V. Smith; pGBT9 and pGAD424 ((34)) from S. Fields; pMa424 (35) from T-H. Chang; PL164 and
PL181 (36) from P. Legrain; pNK1009 (37) from E. Alani and N. Kleckner;
Plasmids were constructed with standard cloning techniques, PCR
amplification (43), or oligonucleotide-directed mutagenesis of single
strand DNA (44). For some plasmid constructions, DNA ends with 5'
overhangs were "blunted" by filling in the overhangs with Klenow.
Constructions involving PCR, mutagenesis, or the insertion of oligos
were confirmed by dideoxy sequencing.
SNP1 Plasmids--
The 1.8-kbp SmaI-HincII
fragment from pJJ215 was ligated into the blunted StyI sites
of plasmid pDi thereby replacing the 500-bp SNP1 fragment to
generate plasmid pPS4 (snp1::HIS3). For constructing pGBT9-SNP1, BamHI and
EcoRI sites were added to the 5' and 3' ends, respectively,
of the SNP1 ORF by PCR using oligos oSR37 and oSR38 and
template plasmid pRS3D. The amplified fragment was cut with
BamHI and EcoRI restriction endonucleases and
then ligated into pGBT9 immediately downstream of the Gal4 DNA binding domain codons 1-147. The SNP1
BamHI-EcoRI insert was subcloned from pGBT9 into
pGAD424 (pGAD-SNP1) and pMA424 (pMA424-SNP1)
downstream of the GAL4 activation domain.
For in vitro expression of FLAG-tagged Snp1p, unique
BglII and HindIIII sites at the 5' and 3' ends of
the SNP1 ORF were introduced by PCR with oligos oSR60 and
oSR61 and template plasmid pRS3D. The resulting DNA fragment was cut
with BglII and HindIII and then subcloned into
the corresponding sites of pGAD424 to create pJD1. Hybridized oligos
oSR68 and oSR69 encoding the FLAG-tag were ligated into the
XhoI and BamHI sites of pJD1 to create plasmid pSR171. The BglII-BamHI SNP1 fragments
from pJD1 and pSR171 were subcloned into the BamHI and
BglII sites of pCite2a to generate plasmids pSR174 and
pSR175, respectively.
EXO84 Plasmids--
The 5.4-kbp SacI-SalI
EXO84 genomic DNA fragment from phage
For creating the exo84 null mutation, the
PstI-SmaI LEU2 fragment from pJJ282
was inserted into the PstI and blunted EcoRI sites of pCM28 to form pCM32
(exo84::LEU2). For constructing
deletion mutations in EXO84 in vitro, a unique
NcoI site was introduced just 3' of the translational stop
codon by mutagenesis of pSA35 with oligo oSA11 to create pSA45. A
unique MluI site just 5' to the translational start codon
was introduced by mutagenesis of pCM29 with oligo oSA10 to create
pSA44. DNA fragments containing the 5' and 3' mutations were used to
replace the corresponding fragments in pCM29 to create pSA46. To create
the exo84-2 mutation, codons 636 and 637 were changed from
AGA-TCT to AGG-CCT in pSA46 with oligo oSA13 to create a unique
StuI site in pSA50. The XcmI-NcoI fragment from pSA50 was subcloned into pSA45 to form pSA53. A stop
codon immediately after codon 636 was then introduced by ligating
hybridized oligos oSA19 and oSA20 into pSA53 DNA cut with
StuI and NcoI to create plasmid pSA55. Plasmid
pSA57 (CEN-HIS3-exo84-2 (Eco47III-NcoI)) was created by subcloning the
BsrGI-NcoI fragment from pSA55 into pSA35.
For expression of the HA-tagged Exo84p in yeast cells, two copies of
the HA tag were inserted at the C-terminal end of Exo84p in three steps
as follows. 1) Unique NheI and AflII sites were introduced just before the stop codon by in vitro
mutagenesis of pSA46 with oligo o46dseM4TAG to create pSA46dse. 2)
Hybridized oligos oHA2TAG-a and oHA2TAG-b were inserted into the
NheI and AflII sites of pSA46dse to create
plasmid pksEXO84HA. 3) The 2-kbp XcmI-NcoI
fragment of pksEXO84-HA was subcloned into the corresponding sites of
pSA57 generating pEXO84-HA2. For expression of Exo84p in
vitro, additional restriction enzyme sites were introduced by
ligating hybridized oligos oSA5 and oSA6 into the PstI and BglII sites of pSR165 to create pSA23. The
NspI-SalI EXO84 fragment was subcloned
into the NspI and SalI sites of pSA23 to create pSA24 (CEN,LEU2,BglII-EXO84); this encodes EXO84
lacking the first 14 codons. The BglII-SalI
fragment containing the EXO84 ORF was subcloned from pSA24
in two steps into the corresponding sites of pCite2a to create pSA40;
this construction added on the first 32 codons of the pCite2a (from
NcoI to BglII sites) plus one codon (ATC) 5' to
codon 15 of EXO84.
PRP8 and Other Plasmids--
The 2.9-kbp,
EcoRV-EcoRI, PRP8 fragment from pJDY13
was subcloned into corresponding sites of pCite2a to create pSR190.
Hybridized oligos oSR95 and oSR96 encoding two copies of the HA tag
were ligated into the NotI and XhoI sites of
pSR190 to generate pSR207. For generating the leu2d595
allele, the blunted ends of the 3.9-kbp BglII-BamHI HisG-URA3-HISG fragment
from pNK1009 were ligated to the EcoRV and blunted
ClaI sites of pJJ283 to generate plasmids pPS9a and pPS9b.
Yeast and Bacterial Strains
Strains Obtained from Others--
Yeast strains HF7c
(MATa, ura3-52, his3-200, ade2-101, lys2-801,
trp1-901, leu2-3, 112, gal4-542, gal80-538,
LYS2::GAL1-HIS3, URA::GAL4
17-CYC1-lacZ) and SFY526 (MATa, ura3-52,
his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542,
gal80-38, URA::GAL1-lacZ) were obtained from
CLONTECH and S. Fields, respectively. Bacterial
strains DH10B (F Yeast Strains Constructed in This Study--
Wild type
haploid yeast strains SRYwt-g and SRYwt-h with the leu2d595
mutation were generated from strains SRYwt-a and SRYwt-d (45) by 1)
targeted integration of the SalI, HisG-URA3-HisG DNA fragment from pPS9, and 2) selection on 5'-FOA for recombinants that had lost URA3 marker from the integrated fragment.
Haploids SRYwt-g and SRYwt-h were mated to create DSR1515
(Mata/
Diploid TSR995-10, heterozygous for the
exo84::LEU2 null mutation, was created
from strain DSR1515 by targeted integration of the
Eco47III-SalI DNA fragment from pCM32. Tetrad
analyses of the sporulated diploid TSR995-10 showed the
EXO84 gene to be essential; only two viable spores per
tetrad were recovered in the 16 tetrads dissected, and none of these
spores were Leu+. However, when plasmid pSA36
(pCEN-URA3-EXO84(Eco47III-NcoI)) was
introduced into TSR995-10, and the resulting transformant TSR1131 was
sporulated and analyzed, viable LEU2 URA3 spore clones were
obtained. Furthermore, these viable TSR1131 spore clones required the
plasmid pSA36 for growth as evidenced by the fact that 5-FOA-resistant
clones were not obtained. TSR995-10 was also transformed with plasmid
pSA18 to create strain TSR1028, sporulated, and dissected to generate
haploid TSR1028-2-13A (Mat Two-hybrid Screens and Assays
HF7c cells were transformed with the pGBT9-Snp1p to obtain the
strain TSR385-1. The pGAD library was then introduced into TSR385-1 by
electroporation as described (32) except that 0.5 µg of single-strand
salmon sperm DNA (average molecular mass of 300 nucleotides) and 1-2
µg of library DNA were added to 400 µl of electrocompetent cells.
Transformants were selected by growth on medium lacking leucine,
tryptophan, and histidine and supplemented with 1 M
sorbitol and either 0, 5, or 20 mM 3-AT. The prototrophs obtained were further screened, if necessary, by replica plating onto
synthetic medium containing 5 or 20 mM 3-AT.
His+ prototrophs which grew in the presence of 20 mM 3-AT were replica-plated onto medium supplemented with
5-bromo-4-chloro-3-indolyl The pGADf library plasmids from these 24 yeast candidates were
recovered from total yeast DNA by electroporation into E. coli BA-1 cells. Transformed bacterial cells were first selected
on LB medium with 100 mg/ml ampicillin and then replica-plated onto M9
medium lacking leucine to detect LEU2-marked pGAD-library
plasmids. The LEU2-marked plasmids were then amplified in
E. coli DH10B cells and sequenced with Primer 1 (CLONTECH, Inc.). Deduced protein sequences were
compared using BLAST, FASTA, and TFAST algorithms (47-49). The
recovered plasmids were then electroporated into strain TSR592-2
(strain SFY526 with the bait plasmid pMA424-BD-SNP1) and
tested again for transcription activation via quantitative In Vitro Transcription, Translation, and
Coimmunoprecipitation
The following plasmids were cut with restriction endonucleases
to produce linear templates for in vitro transcription:
pSR174 and pSR175 with SphI; pSA40 with SalI; and
pSR190 and pSR207 with XhoI. The templates were transcribed
with T7 RNA polymerase and then treated with RQ1 DNase. The RNAs were
purified from the reactions by phenol and CHCl3/isoamyl
alcohol extractions and by ethanol precipitation after which they were
dissolved in water and stored at In vitro translations for coimmunoprecipitation experiments
were combined, supplemented with protease inhibitors (0.1 mg/ml chymostatin, 2 mg/ml aprotinin, 1 mg/ml pepstatin A, 7.2 mg/ml trans-epoxysuccinyl-L-leucylamido-(4-guanidino)
butane, and 0.5 mg/ml leupeptin), and incubated at 30 °C for 30 min.
The total volume was brought up to 250 µl with ice-cold NET-2 (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.05%
Nonidet P-40) and added to either anti-FLAG M2-agarose, anti-HA
16B12-agarose, or the control protein G-agarose. The samples were
slowly rotated at 4 °C for 30 min. The agarose was then washed 4 times with NET-2. Proteins were released from the agarose by heating at
95 °C for 5 min in 50 mM Tris-Cl, pH 8, 200 mM Western Analysis
For the detection of GAL4 BD-SNP1 fusion in TSR385-1,
exponentially growing cells were harvested and resuspended in 250 mM sodium phosphate, pH 7.0, 10 mM EDTA
supplemented with 0.1 mg/ml chymostatin, 2 mg/ml aprotinin, 1 mg/ml
pepstatin A, 7.2 mg/ml trans-epoxysuccinyl-L-leucylamido-(4-guanidino)
butane, and 0.5 mg/ml leupeptin (Buffer A). Cell lysates were prepared
by grinding with glass beads (29). Samples containing 50-150 µg of
protein were fractionated by SDS-PAGE and then transferred onto
Immobilon-P membranes (Millipore) in Towbin buffer containing 0.01%
SDS and no methanol (43). Primary rabbit anti-Gal4p antibody was
diluted 1:2000 in antibody buffer (5% nonfat milk, 10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, and 0.01% Tween 20). The
secondary antibody, sheep anti-rabbit IgG conjugated with horseradish
peroxidase, was diluted 1:3000 in antibody buffer. Proteins were
visualized by chemiluminescence.
Proteins from WCE and extract fractions were analyzed as just described
except that the primary antibodies (anti-HA 12CA5 or 16B12 mAbs, and
anti-FLAG M2 mAb) were diluted 1:3000 and 1:1500, and the secondary
antibody, sheep anti-mouse IgG conjugated with horseradish peroxidase,
was diluted 1:10,000.
RNA Analyses
Total RNA from yeast cells was extracted as described previously
(51). Oligos were radiolabeled at their 5' ends with polynucleotide kinase as described (43) except that PEG8000 was added to
3%. Primer extension reactions for ScR1 and either U5 or Rpl30 RNAs used 25 µg of total RNA per sample and oligos oSR139 and either oSR90
or oSR126 as described (52), except that the reactions were
supplemented with 50 ng/µl actinomycin D. Reactions for actin and
ScR1 RNAs used oligos oSR180 and oSR139 and the conditions of Rymond
et al. (53). The extension products were fractionated by
electrophoresis in 6% polyacrylamide gels in 8 M urea, 89 mM Tris borate, and 2 mM EDTA, pH 8.3 (43), and
measured with a Molecular Dynamics Storm PhosphorImager. The peak areas
of the bands were determined with Molecular Dynamics ImageQuant
software. The 0.5-h samples were not included in our analyses due to
technical difficulties with some samples. The data were analyzed by the Anderson-Darling test for normal distribution and by both the Bartlett
and Levene tests for variance homogeneity in Minitab (Mac version 8).
Differences in the means were analyzed with SAS Institute Statistical
software (version 8.1) using the General Linear Model ANOVA procedure
with repeated measures and least squares means. The Greenhouse-Geisser
adjustment for covariance homogeneity and circularity was used for
multivariate ANOVA (54, 55). In one case (Rpl30 mRNA) where the
variances were not homogeneous (p < 0.01), the
non-parametric Mood's median test (in Minitab) was also used to
compare the 4-h levels in the mutant and wild type strains.
In Vitro Splicing Reactions
The yeast wild type, radiolabeled actin precursor RNA for
splicing assays was synthesized in vitro as described (56).
WCEs were prepared in liquid nitrogen by the method described
previously (56). The fractions 40P3 and 40W were made from yeast
strains TSR1200, TSR1210, and TSR1280, as described previously (57). For testing heat sensitivity in vitro, either nothing,
condition 4 (1.5 mM MgCl2 and 1.5 mM dithiothreitol (58)), or condition 4 plus 1 mM ATP was added to WCE. WCEs were then heated at either 38 or 41 °C for various times and cooled on ice. The conditions for
inactivating and assaying the prp2-1 and prp5-3
WCEs were as described (45, 58) except that the splicing reactions were incubated at 23 °C for 10-15 min. For complementation assays, the
splicing reactions contained 30% (v/v) WCE and either 10% Buffer D
(56) or the indicated amounts of 40P3 or 40W in Buffer D. Splicing
reactions for native gel electrophoresis were analyzed as described
(9, 59).
Prp8p and Exo84p Are Identified as Proteins Interacting with
Snp1p--
To use the two-hybrid system to identify proteins that
interact with Snp1p, we constructed a fusion between the Gal4p DNA binding domain and the entire Snp1p as bait. The resulting fusion protein, BD-Snp1p, was expressed and fully functional in yeast cells
(see "Experimental Procedures"). The plasmid
pGBT9-BD-SNP1 encoding this fusion was then put into a yeast
strain with the HIS3 and lacZ reporter genes for
the initial selection and screen with the two-hybrid system. A library
of yeast genomic DNA fragments fused to the sequence encoding the Gal4p
activation domain (pGADf-library) was then introduced. From a total of
nearly 450,000 transformants containing both bait and library plasmids,
we eventually identified eight library plasmids that activated gene
expression levels from 4- to 424-fold higher than the negative
control (Fig. 1).
Sequencing these eight pGADf library plasmids revealed that they
encoded fragments of four different genes. The USA1 DNA
fragment encoding amino acids 387-443 of ORF YML029W was isolated four times. The plasmid pREP8-5 that gave the highest activity had only
USA1 DNA as the insert, whereas the three other plasmids contained additional insert sequences: pREP5-6 and pREP10-22 had 8 codons derived from at least one other ORF, APC1, at the C
terminus of the fusion; and pREP9-1, had an in-frame fragment upstream of USA1. The same USA2 fragment was isolated
twice and encodes 28 amino acids (1166-1193) of the 280-kDa Prp8
protein. The two USA2/PRP8 plasmids differed in that
pREP5-16R had an additional, in-frame DNA fragment encoding part of
pyruvate carboxylase. The USA3 gene fragment in pREP5-7
encodes 52 amino acids (506) of ORF YBR102C that has
recently been identified as EXO84 (28). The last plasmid,
pREP7-2, encodes amino acids 243-262 of chitin synthase 1.
Our two-hybrid screen identified one known splicing factor, Prp8p,
which is a very large, highly conserved, essential protein subunit of
the spliceosomal U5 snRNP (60). Two proteins, Usa1p and Exo84p, were
potentially new splicing factors. Exo84p has only been recently
identified as a conserved subunit of the exocyst complex involved in
the last steps of the secretory pathway (28). The exocyst complex is
important for membrane and cell wall synthesis as well as protein
secretion (61). Nonetheless, Exo84p is also a good candidate to be a
splicing factor. It is essential for mitotic cell growth (see
"Experimental Procedures" and Ref. 62), and it has some predicted
protein motifs relevant to splicing. There are three potential nuclear
localization signals (7KKARNNWKHVKK,
62RERSRVATSMQRR, and 484NKNKRR), and at least
one nuclear export sequence (131LNLSTADLSL) (63). Two
S(R/R)E dipeptides repeats (62RERSR and
519SRPRSR) especially fit the consensus target site
(R(R/E/D/P)(R/H)S(R/P)) of SR protein kinases (64). The second of these
SR repeat sequences lies in the same region (amino acids 506-557) that
interacts with Snp1p in the two-hybrid assay. A "PatMatch" search
of all yeast ORFS in the Saccharomyces Gene Data base (65)
showed that these two SR repeat sequences occur in only 4 and 7 ORFS,
respectively, including YBR102C/EXO84. Finally, two regions
(amino acids 226-278 and 542-584) with heptad repeats of potential
amphipathic helices of coiled-coils have also been noted recently by
others (28). Such coiled-coils may mediate protein-protein interactions
or nucleic acid binding (66). The potential of Exo84p as a splicing factor led us to test its function in pre-mRNA splicing as
described below.
The other protein, Usa1p, has no essential function in mitotically
growing yeast cells (data not shown; Ref. 67) nor could we discern a
function in pre-mRNA splicing in vivo (data not shown). Predicted to be 97.6 kDa, Usa1p has no obvious homology to any ORF in
the data bases to date. Its only remarkable feature as noted in the
Prosite data base (68) is a ubiquitin-like domain encoded by amino
acids 259-318.
The fourth protein, Usa4/Chs1p, synthesizes chitin for closing the
daughter cell wall after cytokinesis (69). Perhaps the interaction
between Chs1p and Snp1p is physiologically relevant. Another
possibility, however, is that normally the Chs1p-Snp1p interaction
would not occur in the cell because the region of Chs1p with which
Snp1p interacts is not exposed or the two proteins do not colocalize.
Snp1p Physically Associates with Prp8p and Exo84p--
To
investigate further the interactions of Snp1p with Prp8p and Exo84p, we
tested if the proteins physically associate by an immunological assay.
The entire SNP1 and EXO84 coding regions were
subcloned for expression in vitro. The FLAG epitope tag was also added to the C-terminal end of Snp1p. Because the large size of
Prp8p precludes efficient expression in vitro, a DNA
fragment encoding a 115-kDa fragment of Prp8p (Prp8f) containing the
Snp1p interaction region was used. Prp8f was also engineered with the HA tag at its C-terminal end. Radiolabeled forms of Snp1p, Exo84p, and
Prp8f were obtained by in vitro translation in the presence of [35S]methionine (Fig.
2). Although the predominant form of
Exo84p in the translation was 24 kDa smaller than the predicted
molecular mass of 85.5 kDa, the ~61-kDa fragment still
contained the Snp1-interacting region and was probably derived from the
full-length protein by cleavage near the N terminus (data not shown).
The individual translations were treated with RNase A and then
incubated together at 30 °C for 30 min after which they were
subjected to immunoprecipitation with either anti-FLAG or anti-HA
monoclonal antibody.
We obtained coimmunoprecipitation for both combinations of proteins.
Snp1p coprecipitated when it was incubated with HA-tagged Prp8pf and
anti-HA antibody (Fig. 2, lane 12). Similarly, Exo84p coprecipitated when it was incubated together with FLAG-tagged Snp1p
and anti-FLAG antibody (lane 19). As controls, the HA-tagged Prp8pf alone precipitated with anti-HA antibody (lanes 11),
but little or none of it precipitated in the absence of antibody
(lane 9) or the HA-tag (data not shown). Little or no Snp1p
precipitated in the absence of HA-tagged Prp8pf (lane 13) or
antibody (lane 10). Similarly, little or no Exo84p
precipitated in the absence of FLAG-tagged Snp1p (lanes 16)
or anti-FLAG antibody (lane 17). Snp1p did not precipitate
with anti-FLAG antibody unless it had the FLAG tag (data not shown).
Both coprecipitations (Snp1p with Prp8f and Exo84p with Snp1p) required
the proteins to be incubated together at 30 °C for at least 15 min
(data not shown). Neither coprecipitation depended on RNA binding as
the individual translations were treated with RNase A before they were
mixed together. Furthermore, the coprecipitations likely do not depend
on most other splicing factors because reticulocyte lysates do not
complement several yeast prp mutant extracts in
vitro (70, 71), an indication that the lysate lacks several
proteins that could functionally interact with the yeast spliceosome.
We conclude that Snp1p does physically associate with Prp8p or Exo84p
in the absence of RNA and most other yeast splicing factors.
A Ts exo84 Mutation Causes Increased Percentages of Pre-mRNA
for Some Transcripts at the Non-permissive Temperature--
Often
proteins that physically associate with one another function in the
same biological process. To determine whether Exo84p functions in
splicing, we first engineered some exo84 deletion mutations
in vitro using convenient restriction endonuclease sites to
delete N- and C-terminal portions. The mutations were then introduced
into yeast cells by the plasmid shuffling method to test their effects
in vivo (29). One of these, exo84-2, is recessive and results in Ts mitotic growth; mutant cells in the asynchronous population either did not divide or divided only one to a few times
after being shifted to 37 °C (Fig. 3).
The mutant grew more slowly than the wild type at lower temperatures
(Fig. 3) and also showed cold-sensitive growth arrest at 16 °C (data
not shown). The exo84-2 mutation substitutes arginine 636 with proline and introduces a stop codon immediately thereafter such
that the mutant protein also lacks the last 117 amino acids of its
C-terminal end. The other deletion mutations and their phenotypic
effects will be described elsewhere.
We next analyzed the transcripts of the intron-containing
RPL30 and ACT genes and the intron-less
SNR5 and SCR1 genes in the wild type and
exo84-2 mutant strains. The yeast strains were grown to
midlog phase at the permissive temperature (26 °C) and then shifted
to the non-permissive temperature (37 °C). RNAs were extracted from
the yeast cells incubated at 26 and 37 °C and analyzed by primer
extension assays. We found that the levels of Rpl30 and actin mRNAs
decreased in the exo84-2 mutant after the shift to 37 °C
(Fig. 4, A, B, and
D); the levels of Rpl30 and actin mRNAs went from 100%
at 0 h to 36 and 40%, respectively, at 4 h. The levels of
both the Rpl30 and actin mRNAs in the mutant were statistically significantly less than the wild type levels during incubation at
37 °C (p < 0.01 and p < 0.05 respectively). Although there were some differences in Rpl30 mRNA
levels among the wild type isolates during the temperature shift (one
isolate (Fig. 4A) showed less of a heat shock response than
the other two (Fig. 4D)), the levels of Rpl30 mRNA were
consistently lower in the exo84-2 mutant than in the wild
type. The prp5-3 mutant, with a known defect in pre-mRNA
splicing (45), also showed marked decreases in Rpl30 and actin
mRNAs at 37 °C (Fig. 4), from 100% at 23 °C to 3% at 37 °C for Rpl30 and from 100% at 26 °C to 17% at 37 °C for
actin. Importantly, when compared with wild type during the incubation at 37 °C, the exo84-2 mutant showed significant increases
in the percent pre-mRNA (Fig. 5) for
the Rpl30 and actin transcripts (p < 0.02 and
p < 0.03, respectively). By 4 h there were 27%
Rpl30 and 28% actin pre-mRNAs in the exo84-2 mutant
compared with 15 and 7% in the wild type. Additionally, the mutant
showed a significantly (p < 0.01) increased rate of
accumulation of percent pre-mRNA for the actin transcript relative
to the wild type during incubation at 37 °C. In contrast, the levels
of the intron-less U5 and scR1 RNAs did not change significantly at
37 °C when comparing levels either between the wild type and
exo84-2 mutant strains or within each strain at 26 and
37 °C (Fig. 4). When the exo84-2 mutant was compared with
the prp5-3 mutant, similar effects on RNA expression were
seen (Fig. 4) except that the prp5-3 mutant showed higher percentages of pre-mRNA compared with wild type at both permissive and non-permissive temperatures, from 22% at 26 °C to 58% for 4 h at 37 °C for Rpl30 and from 49 to 73% for actin. Although the prototypic splicing defective phenotype is described as an absolute
increase of pre-mRNA or splicing intermediates in a mutant compared
with the wild type (see for example Ref. 51), some known splicing
defective mutations (72, 73) such as prp5-3 show effects
like exo84-2, a decrease in the levels of both pre-mRNA and spliced mRNA but an increase in the percent pre-mRNA. We
conclude that the increase in percent pre-mRNA as well as the
decrease in spliced mRNA for the Rpl30 and actin transcripts in the
exo84-2 mutant suggests that the exo84-2 mutation
causes a defect in the splicing of pre-mRNAs or in some other
intron-dependent function at the non-permissive
temperature. However, other effects such as those involving
transcription initiation or mRNA stability may also contribute to
the decreased levels of total Rpl30 and actin transcripts.
The exo84-2 Mutation Inhibits Splicing Activity in Vitro--
If
the exo84-2 mutation directly affects splicing, it should
cause a splicing defect in vitro. To test this, we utilized
the Ts phenotype of the exo84-2 mutation. Previously it has
been shown that a Ts mutation in a splicing factor frequently confers
heat or cold sensitivity for splicing activity in vitro (58,
74). We therefore made active splicing whole cell extracts (WCEs) from the isogenic wild type and exo84 mutant cells grown at the
permissive temperature and then tested these extracts for heat
sensitivity in vitro at different temperatures. The mutant
WCE was inactivated for splicing by short heat treatments at 41 °C
that did not inactivate wild type WCE (Fig.
6A). When radiolabeled actin
pre-mRNA was added to the inactivated WCE, no splicing
intermediates or products were formed. Addition of magnesium to
exo84-2 WCE shortened the incubation period required for
inactivation, but ATP had no effect (data not shown). These results
suggest that the defect caused by the short heat treatments of the
exo84-2 mutant WCE is due to the Ts exo84-2
mutation and occurs before the first splicing reaction.
That the heat sensitivity for the mutant extract is due to the
exo84-2 mutation was assayed in two ways. First, we
determined if an inactivated exo84-2 mutant WCE would
complement inactivated WCEs with some Ts prp mutations in
known splicing factors. If a distinct component is inactivated in each
extract, then different extracts should complement one another. If the
inactivated components are the same or do not readily exchange, then
the extracts will fail to complement. WCE from exo84-2,
prp2-1, prp5-3, and prp9-1 mutants were
individually heated and then mixed pairwise and assayed for splicing
activity at 23 °C (Fig. 6B). Each heated extract had
little or no splicing activity but complemented each of the other
extracts. This result indicates that the heat sensitivity of each
extract is due to the inactivation of specific, different, exchangeable
components in each extract.
Second, we tested if splicing activity of the inactivated
exo84-2 mutant WCE could be restored by complementation with
two fractions (40P3 and 40W) made from wild type WCE. Each extract fraction, 40P3 and 40W, has been shown previously to be enriched for a
subset of splicing factors but inactive for splicing (57). Splicing
activity can be restored when the two fractions are mixed together. To
detect Exo84p in WCE and extract fractions, we created an HA-tagged
form of Exo84p in vitro and substituted it for the wild type
protein in vivo. We found that the fraction 40P3, which contains about 10% of the total protein in the original WCE, was enriched for HA-tagged Exo84p about 5-fold relative to WCE (Fig. 7A). Moreover, the 40P3
fraction with either HA-tagged Exo84p (not shown) or untagged Exo84p
complemented the heat-inactivated exo84-2 mutant WCE for
splicing (Fig. 7B). The other fraction, 40W, was deficient
in Exo84p (Fig. 7A) and did not restore splicing activity to
inactivated exo84-2 WCE (Fig. 7B). Additionally,
we made the 40P3 extract fraction from the exo84-2 mutant
and tested its heat sensitivity and complementing activity in
vitro. When exo84-2 mutant WCE and mutant 40P3 fraction
were heated together, splicing activity was lost. When mutant WCE and
wild type 40P3 were heated together, however, splicing activity was
retained (data not shown). These results indicate that mutant Exo84p in the 40P3 fraction is also heat-sensitive in vitro and that
this sensitivity is recessive in vitro as it is in
vivo. The collective results from all these in vitro
splicing assays are consistent with the exo84-2 mutation
having a direct effect on splicing.
The exo84-2 Mutation Inhibits an Early Step in Spliceosome
Development--
The results of the in vitro splicing
assays indicate that the mutation inhibits splicing before the first
catalytic splicing reaction, or even earlier, during assembly of the
spliceosome onto the pre-mRNA. We therefore assayed spliceosome
formation in isogenic exo84-2 mutant and wild type WCEs at
23 °C before and after heat inactivation at 41 °C for 7 min
in vitro. The spliceosome assembly assay uses radiolabeled
actin pre-mRNA as substrate and polyacrylamide gel electrophoresis
under nondenaturing conditions to separate the complexes formed on the
substrate (9). These complexes normally migrate as three bands,
The interaction of Exo84p with Snp1p, a component of the U1 snRNP,
suggests that Exo84p associates with the U1 snRNP. We assayed for such
an association by in vitro immunoprecipitation assays using
WCE with HA-tagged Exo84p, and immobilized anti-HA antibody. Any
spliceosomal snRNAs coprecipitating with HA-tagged Exo84p were detected
by primer extension using radiolabeled oligos specific for each
spliceosomal snRNA. We were unable to detect an association of Exo84p
with any of the five snRNAs, even under low salt stringency (50 mM NaCl) (data not shown). Similarly, we could not detect an association of Exo84p with radiolabeled pre-mRNA added to the extract (data not shown). Therefore, either Exo84p does not associate with a spliceosomal snRNP or pre-mRNA or the assay failed to
detect the association.
In this study we found two proteins to associate physically with
Snp1p, a component of the yeast spliceosomal U1 snRNP. One of the
proteins, Prp8p, is a known spliceosomal component essential for
splicing. The other, Exo84p, has recently been found by others (28) to
be essential and required for the secretory pathway. We have discovered
that Exo84p is also involved in pre-mRNA splicing. An
exo84-2 mutation engineered in vitro was found to
be Ts for mitotic growth in vivo and to alter expression of
two genes containing introns. The exo84-2 mutation, like the
prp5-3 mutation in a known splicing factor, increased the
levels of actin and RPL30 pre-mRNA relative to the spliced mRNA
at the non-permissive temperature. In vitro assays indicated
that Exo84p plays a direct role in splicing in that inactivation of the
protein in vitro resulted in a loss of splicing activity.
When the Ts exo84-2 mutant extract was heat-treated in
vitro, it became defective for splicing and prespliceosome formation. These results suggest that Exo84p could be a splicing factor, a factor that modifies a splicing factor, or both.
The Interaction between Prp8p and Snp1p Suggests New Roles for
Snp1p--
Although it has been conjectured earlier that the U1 snRNP
leaves the spliceosome before the tri-snRNP binds, our finding that
Snp1p interacts with Prp8p strongly suggests that the U1 snRNP is
acting at additional, later steps in the pre-mRNA splicing pathway.
Prp8p is a very large protein component of the U5 snRNP that is
required for several steps in the pre-mRNA splicing pathway (75,
76). It is necessary for the U4/U6.U5 tri-snRNP to form and bind to the
prespliceosome. It is essential for both the first and second
transesterification reactions of splicing during which it can be
cross-linked to both the 5'- and 3'-SS. It is highly conserved, sharing
65 and 61% identity with the human and worm Prp8 proteins throughout
nearly its entire length of 2400 amino acids (60, 77). Yet, Prp8p
appears to be relatively featureless, others have noted only predicted
amphipathic helices (amino acids 643-699 and 1626-1651) (78) and a
non-conserved, proline-rich region at the N terminus (60).
The 28 amino acids (1166-1193) of Prp8p that associate with Snp1p in
the two-hybrid assay (Fig. 1) delineate a very small, charged region in
the middle of Prp8p. Remarkably, substitution mutations in some of
these amino acids (1191-1194) also suppress the cold-sensitive U4Cs1
mutation in the U4 snRNA (78). U4cs1 extends the base pairing of the U4
and U6 snRNAs in the U4/U6 snRNP. This extension occludes the ACAGA box
of U6 snRNA (the region which normally pairs with the 5'-SS during
catalysis) and enhances the stability of the U4/U6 duplex. Because of
these and other genetic interactions, Kuhn and Brow (10) suggested that Prp8p may also coordinate unwinding of the U4/U6 snRNAs with unwinding of the U1/5'-SS duplex during spliceosome activation to catalytic competency. The Snp1p-Prp8p interaction could also be involved in both
unwinding the U4/U6 snRNAs and destabilizing the U1/5'-SS duplex and in
coordinating these two events. The 70K protein, the human homolog of
Snp1p, interacts with the U1 C protein which stabilizes the U1/5'-SS
duplex (13). Both the yeast U1 Snp1p and C proteins contact the 5'-SS
(6). Recently, the yeast C protein has also been found to counteract
Prp28p, a helicase that disrupts U1/5'-SS stability (12). Therefore, it
seems likely the yeast Snp1p and C proteins also interact and that this
interaction would help stabilize the U1/5'-SS duplex. Furthermore, the
Snp1p-Prp8p interaction might antagonize the Snp1p-C interaction and
help to destabilize the duplex. This function of the Snp1p-Prp8p
interaction is consistent with observations that about the time the
tri-snRNP complex containing Prp8p becomes part of the spliceosome, the U1 snRNP becomes less stably associated with the spliceosome (9, 11).
Snp1p may also physically contact Prp44p (Brr2/Rss1/Slt22/Snu246p) (79). Prp44p is now thought to be the most likely DEAD box helicase to
unwind the paired U4/U6 snRNAs (80).
It is also possible that the Snp1p-Prp8p interaction functions in
docking the U4/U5.U6 tri-snRNP onto the pre-spliceosome and, more
specifically, in guiding Prp8p to the 5'-SS region. Consistent with
this possibility, the U1 and U5 snRNPs are both present in at least one
spliceosomal complex during spliceosome development (9, 11). Moreover,
U1/U5 snRNP complexes have been observed in both yeast (9) and human
(81) splicing extracts, and the human U1 and U5 snRNAs can be
cross-linked. Finally, there is the possibility to consider that Snp1p
has a function involving Prp8p that is independent of the U1 snRNP. For
example, the 70K protein shuttles between the nucleus and cytoplasm
without U1 snRNA (82).
Role of Exo84p in Pre-mRNA Splicing--
We have shown here
that exo84-2 mutant extracts are heat-sensitive for splicing
in vitro. Several lines of evidence support our conclusion
that this sensitivity is due to inactivation of the exo84-2
mutant protein in vitro, thereby indicating that Exo84p has
a direct role in splicing. The inactivated exo84-2 extract complemented inactivated splicing extracts with defects in other splicing factors. Moreover, the inactivated exo84-2 extract
was complemented by the extract fraction 40P3 that was enriched for wild type Exo84p, and the 40P3 fraction made from the
exo84-2 mutant was heat-sensitive in vitro. The
complementation patterns of the inactivated exo84-2 mutant
extract with different mutant and wild type extracts and extract
fractions are consistent with the recessive nature of the
exo84-2 mutation in vivo and a loss of Exo84p
function. However, we were not able to restore activity to the mutant
extract by adding back a GST-Exo84 fusion protein isolated from yeast
cells2 and therefore cannot
exclude the possibility that the mutation indirectly causes a Ts defect
in splicing.
We think the possibility that the Ts defect is indirect is unlikely for
two reasons. First, the heat sensitivity of the mutant 40P3 fraction
argues against the idea that the temperature sensitivity in
vivo and in vitro is due to the indirect effect of a
decreased concentration of an essential splicing factor. Second, the
number and types of interactions detected among Exo84p and some other splicing factors reveal a functionally congruent pattern. Interactions between Exo84p and Snp1p (this study), Exo84p and Prp40p, another U1
snRNP-specific protein,3
Prp8p and Prp40p (7), and Snp1p and Prp8p (this study) have been
detected. Exo84p itself may also interact with the U5 snRNP as a
two-hybrid study identified three clones interacting with Prp8p that
encode amino acids 615-753 of Exo84p (78). Interestingly, most of
these Exo84p amino acids are deleted in the exo84-2
mutation. When the interactions between the U1 and U5 snRNPs as
discussed above are considered as well, the pattern of interactions
suggests that Exo84p physically interacts with the spliceosome or at
least the U1 snRNP. It therefore seems more likely that the inability of the purified GST-Exo84 fusion protein to complement the inactivated extract is due to (a) the altered activity of fusion or
(b) the absence of a required, Exo84p-associating factor
that is not readily exchangeable in the inactivated exo84-2
extract. Such a factor would probably not be Prp2p, Prp5p, or Prp9p
because the corresponding inactivated mutant extract readily
complemented the inactivated exo84-2 extract in
vitro. In conclusion, the in vitro and in
vivo results collectively indicate that the heat sensitivity of
the mutant exo84-2 extract is due to a loss of function of
mutant exo84-2p. The simplest explanation for the in vitro
complementation pattern is that normally wild type Exo84p has a direct
role in splicing.
Inactivation of mutant exo84-2p prevents prespliceosome formation
in vitro (Fig. 8). This defect is compatible with the
physical interaction of wild type Exo84p with Snp1p. Yeast
prespliceosome formation requires an intact U1 snRNP bound correctly to
the pre-mRNA in the
The in vivo assays showed 1) that the levels of spliced
mRNAs decreased and 2) that the percentage of transcript as
unspliced pre-mRNA increased for the Rpl30 and actin transcripts at
37 °C in the exo84-2 mutant. The percent pre-mRNAs
for the RPL30 and actin transcripts were 3.8- and 3-fold, respectively,
that at 26 °C in the mutant, as well as 2- and 4-fold, respectively,
the percent pre-mRNA in the wild type strain at 37 °C. These
in vivo effects are relatively mild compared with the
in vitro effects on splicing. As our in vitro
data indicate a direct role for Exo84p in splicing, why did we not see
a larger accumulation of pre-mRNA in the exo84-2 mutant
cells in vivo? Like mutations in some other splicing factors
(86), the exo84-2 mutation is more severe in vitro than in vivo. The severity of a Ts mutation
in vivo can depend on the genetic background of a strain
(58). Furthermore, several RNA processing pathways can influence the
turnover of RNAs in vivo and could lower the percent
pre-mRNA (87-90). Conversely, the exo84-2 mutation
could also affect other intron-dependent processes in
addition to splicing that modulate RNA levels in vivo. We
would not have detected these processes in our in vitro splicing assays.
The decreased levels of Rpl30 and actin RNAs seen in vivo
could also be part of a stress response provoked by interrupting the
secretory pathway (91). Several Ts sec mutations conferring defects in different steps in the secretory pathway also lead to
repression of transcription of the ribosomal RNA and protein genes but
not other genes (92, 93). To our knowledge, no mutations in the exocyst
subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84
proteins (28)) have been tested for this phenomenon. However, the
effects of the exo84-2 mutation on actin RNA levels suggest
that there could be some differences in the response invoked by this
mutation compared with the sec mutations that have been examined. We observed in this study that both the splicing and total
level of actin RNAs decreased as a result of the exo84-2 mutation. Warner and colleagues (92) have found that actin mRNA levels are unaffected in the non-exocyst sec mutants.
Furthermore, these sec mutations act earlier in the pathway
than when the exocyst functions. It will thus be interesting to see
whether the exo84-2 mutation also causes decreased levels of
non-intron-containing, ribosomal protein gene RNAs.
A Dual Role for Exo84p in Secretion and Pre-mRNA
Splicing?--
Most of the secretory pathway in yeast is dedicated to
the synthesis of cellular membranes and the secretion of proteins for the cell wall (96). As part of the exocyst complex, Exo84p tethers secretory vesicles onto the plasma membrane (28). These vesicles contain the proteins and lipids necessary for membrane expansion, as
well secreted proteins such as invertase. Perhaps Exo84p has two
distinct functions in the cell, one for secretion and one for
pre-mRNA splicing. Alternatively, the activity of Exo84p in secretion may be somehow functionally linked to pre-mRNA splicing. The effect, if any, of the exo84-2 mutation on secretion
will thus be interesting to test.
There have already been some genetic hints of interactions between
pre-mRNA splicing and the secretory pathway in S. cerevisiae. A mutation in LUC7, which encodes another
U1-snRNP-specific protein essential for splicing (72), also leads to
enhanced export of secreted proteins (97). A mutation in
SLT11, which is essential for splicing in vivo
and in vitro, also causes aberrant cell wall synthesis or
structure (98). In the case of the slt11 mutation, however,
the effect on secretion may be indirect; slt11 reduces splicing of Sar1 pre-mRNA that encodes a protein essential for endoplasmic reticulum-to-Golgi trafficking (99). Finally, several proteins involved in vesicular transport in addition to Exo84p have
been found to interact with at least 10 splicing factors in two-hybrid
screens (79, 100). Although some of these two-hybrid interactions may
not be physiologically relevant, the number of interactions between
components of the secretory pathway and spliceosomal proteins suggest
that there could be an extensive network linking splicing and secretion.
Exo84p could have a regulatory role for splicing by linking the
functional state of the secretory pathway to pre-mRNA splicing. For
example, in yeast cells the stress of accumulated, unfolded proteins in
the endoplasmic reticulum induces Hac1 RNA splicing, although this
splicing involves non-spliceosomal factors (94). In mammalian pituitary
cells, depolarization leads to changes in the alternative splicing of
some ion channel pre-mRNAs via a
Ca2+/calmodulin-dependent protein kinase
(95).
When and Where Do Exo84p and Snp1p Interact in the Yeast
Cell?--
Finding that Exo84p interacts with Snp1p and that it is
required for splicing in vitro raises the questions of when
and where Exo84p and Snp1p interact in the yeast cell. A number of
scenarios can be considered at this point. Because pre-mRNA
splicing occurs in the nucleus, the simplest hypothesis is that Exo84p
interacts there with the spliceosomal components. However, Exo84p, as
part of the exocyst complex, has been shown to localize to regions of
plasma membrane expansion and cell well synthesis (28). Nonetheless, there is a significant pool of free Exo84p that does not cosediment with the exocyst complex. We have noted that Exo84p has possible sequences for nuclear localization and for nuclear export, and thus it
may shuttle between the nucleus and cytoplasm. Npl3p, another protein
that may interact with the U1 snRNP (101), also shuttles between the
nucleus and cytoplasm (102). Interestingly, nuclear import of Npl3p is
regulated in part by the cytoplasmic Sky1 kinase, a conserved SR
protein kinase (64). Sky1p phosphorylates a single serine within a
consensus target site on Npl3p. Exo84p has two putative copies of this site.
Another possibility is that Exo84p interacts with splicing factors
while they are in the cytoplasm. In mammalian cells, the snRNPs are
assembled in the cytoplasm, and the mature snRNPs are then transported
to the nucleus (103). Additionally, some snRNP factors, like the 70K
protein, may shuttle between the nucleus and cytoplasm independently of
their snRNP partners (82). Finally, Snp1p, or even the U1 snRNP, could
also relocate to the cytoplasm as part of a stress response. Some yeast
Ts sec mutations that interrupt secretion and induce
repression of transcription of ribosomal protein genes also cause
relocation of nucleolar and nucleoplasmic proteins, such as Npl3p and
some U1 snRNP proteins, to the cytoplasm (104).
By whatever mode Snp1p and Exo84p come together, their interaction
links RNA processing to the secretory pathway. The link may be simply
that Exo84p has two independent functions in the cell. Alternatively,
Exo84p and Snp1p may be components of a larger regulatory network
comodulating the two processes. In either case, this link may provide
one of only a few examples discovered in yeast of the regulation of
splicing in response to a physiological stimulus.
We thank the following for plasmids,
antibodies, and strains: E. Alani, J. Beggs, T-H. Chang, S. Fields, J. Hopper, N. Kleckner, P. Legrain, V. Lundblad, A. Murray, V. Smith, D. Weist, and M. Werner-Washburne. We thank the following for advice: C. Wheeler on DNA sequencing; D. Peabody and J. Summers on mutagenesis; G. Brock, L. Salter, and an anonymous reviewer on statistics; and K. Pruter on computer analyses of Exo84p. We thank D. A. Brow and
M. A. Osley for comments on the manuscript; T. Chang, A. Kuhn, D. Brow, and P. Siliciano for communicating results prior to publication; and the University of New Mexico Center for Genetics in Medicine for
oligo synthesis and some DNA sequencing.
*
This work was supported by National Science Foundation
Grants MCB9219408 and MCB9709915 and by grants from the Dedicated
Health Research Funds Committee of the University of New Mexico Health Sciences Center.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: Dept. of Microbiology, University of Pennsylvania
School of Medicine, 421 Curie Blvd., BRB2-3, Philadelphia, PA 19104.
¶
Current address: Dept. of of Molecular Genetics, Harvard
Medical School, Massachusetts General Hospital, Charlestown, MA 02129.
**
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Microbiology, University of New Mexico Health Sciences
Center, 900 Camino de Salud, NE, Albuquerque, NM 87131. Tel.:
505-272-5830; Fax: 505-272-8199; E-mail: sruby@unm.edu.
Published, JBC Papers in Press, June 25, 2001, DOI 10.1074/jbc.M100022200
2
S. Ruby, unpublished data.
3
P. Siliciano, personal communication.
The abbreviations used are:
snRNP, small nuclear
ribonucleoprotein;
3-AT, 3-amino-1,2,4-triazole;
5-FOA, 5'-fluoroorotic
acid;
ANOVA, analysis of variance;
bp, base pair;
HA, hemagglutinin
epitope;
Klenow, Klenow fragment of E. coli DNA polymerase;
oligo, oligodeoxynucleotide;
ORF, open reading frame;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
PEG, polyethylene glycol;
snRNA, small nuclear RNA;
Ts, temperature-sensitive;
WCE, yeast whole cell splicing extract;
kbp, kilobase pair;
5'-SS, 5'-splice site.
New Roles for the Snp1 and Exo84 Proteins in Yeast Pre-mRNA
Splicing*
§,¶,,
, and**
Department of Molecular Genetics and
Microbiology, University of New Mexico Health Sciences Center,
Cancer Research and Treatment Center,
Albuquerque, New Mexico 87131
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage clone 70362 and cosmid clone 8337 with USA1
and EXO84, respectively, from the American Type Culture Collection; pCite2a from D. Weist; and pJDY13 (38) from J. Beggs. The
pGADF library of Gal4p activation domain fusions (YL4000AF) was made by
CLONTECH from Sau3AI-cut, SFY526 genomic
DNA inserted into the BamHI site of three plasmids, pGAD1F,
pGAD2F, and pGAD3F (39). The pRS300 and pRS400 series of yeast shuttle
vectors (40), integrating vector Yiplac211 (41), and plasmids pJJ215, pJJ282, and pJJ283 (42) were as described previously.
was subcloned into
plasmids pKSII+ and pUC119 to create pCM28 and pCM29, respectively. The
Eco47III-SalI fragment containing EXO84 and 610 and 80 bp of its 5'- and 3'-flanking sequences, respectively, was
subcloned from pCM29 into pRS316 and pRS313 to create pSA18
(CEN-URA3-EXO84(Eco47III-SalI)) and
pSA20
(CEN-HIS3-EXO84(Eco47III-SalI)). Because of the small amount of sequence from the EXO84 stop
codon to the SalI site, we subcloned additional 3'-flanking
DNA from a wild type yeast strain. For this purpose we constructed the plasmid pSA26 (HIS3-BglII-exo84) containing ~600 bp of the
N terminus of EXO84 with a BglII site just 5' to
EXO84 codon 1. Plasmid pSA26 was target-integrated into
genomic DNA at the EXO84 locus. The plasmid pSA31 with 480 bp downstream of the EXO84 translational stop codon was then
recovered by cutting the yeast DNA with NcoI, ligating the
DNA under dilute conditions to circularize the plasmid DNA, and
amplifying the plasmid in Escherichia coli. The
XcmI-NcoI fragment encoding the 3' end of
EXO84 was subcloned from pSA31 into the XcmI and
blunted SalI sites of pSA20 to create pSA35 (CEN-HIS3-EXO84(Eco47III-NcoI)).
Similarly, the ClaI-NcoI fragment from pSA31 was
subcloned into the ClaI and blunted SalI sites of
pSA20 to create pSA36 (CEN-URA3-EXO84
(Eco47III-NcoI)).
, mcrA 
(mrr-hsdRMS-mcrBC)
80dlacZDM15, lacX74,
deoR, recA1, endA1,
araD139,(ara, leu)7697,
galU, galK, -, rpsL,
nupG) and BA-1 (thr1, leuB6,
thi1, thyA, trpC1117,
hsrK, hsmK,
strr, hisB, tetr) were
obtained from Life Technologies, Inc., and A. Murray, respectively.
, his3d200/his3d200, HIS7/his7,
leu2d595/leu2d595, ura3-52/ura3-52). TSR432
(MATa/a,
SNP1/snp1::HIS3, his3d200/his3d200, HIS7/his7,
ura3-52/ura3-52, leu2d595/leu2d595) was generated from DRS1515 by
targeted integration of the EcoRV-SphI
(snp1::HIS3) DNA fragment from plasmid
pPS4. For testing the function and expression of the
GAL4-SNP1 fusion, TSR432 was then transformed with plasmid pGAD-SNP1, and the resulting diploid, DRP432-8, was
sporulated. Dissected tetrads from sporulated diploids TSR432 and
DRP432-8 were analyzed for germination and growth on medium with
glucose. No snp1::HIS3 spores from 16 TSR432 tetrads were viable, but most snp1::HIS3, pGAD-SNP1 spores
from 12 DRP432-8 tetrads were viable.
, his3d200, leu2d595, ura3-52,
exo84::LEU2, pSA18). Diploid DSR1624 was
created by mating TSR1028-2-13A with wild type DSR1124-2-4D
(Mata, cup1d, leu2-3 -112, his3d200, trp1d,
ura3-52). Haploid DSR1624-2-11B (Mata,
cup1d, his3d200, leu2d595, trp1d, exo84::LEU2,
pSA18) was obtained by tetrad dissection. Strains TSR1200,
TSR1210, and TSR1280 were obtained by transforming haploid
DSR1624-2-11B with plasmids pSA55, pSA35, and pEXO84-HA2, respectively,
and by subsequent selection on 5-FOA at 30 °C.
-D-galactopyranoside (X-gal)
to detect -galactosidase activity. The
His+,lacZ+ candidate transformants were then
grown in liquid selective media at 30 °C to midlog phase and assayed
quantitatively for -galactosidase activity by the glass bead method
(46). Units of activity were calculated as units = (A420 × 1000)/(min × A600 assayed). We obtained 24 candidates with
-galactosidase activities of 0.35 units and above.
-galactosidase assays. The combination of the strain (SFY526) and
vector (pMA424) often gives higher -galactosidase activities than
the strain (HF7C) and vector (pGBT9) used in the initial screen
(50).
70 °C. RNAs were added to
in vitro translation reactions with [35S]methionine and micrococcal nuclease-treated rabbit
reticulocyte lysate according to Promega's conditions (1992 bulletin
TM232). After the reactions were incubated for 30-60 min at 37 °C,
they were treated with RNase A for 30 min. In vitro
translation products were separated by SDS-PAGE (43) and visualized by
fluorography and autoradiography.
-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, and
10% glycerol, separated by SDS-PAGE, and visualized as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
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Fig. 1.
Snp1p-Usap interactions detected by the
two-hybrid transcription activation assay. The -galactosidase
activities for plasmids encoding productive, activation domain
(AD)-USA fusions that interact with the binding
domain fusion to Snp1p (BD-SNP1) are shown. After
being identified in the initial screen and selection (see
"Experimental Procedures"), the AD-USA plasmids were
reintroduced into yeast strain, SFY526, with the vector pMA424 with
either the BD-SNP1 insert (hatched columns) or no
insert (black columns). The mean (±S.D.) -galactosidase
activity of two transformants is shown. The Prp21p-Prp9p interaction
was used as a positive control (open column).
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Fig. 2.
Coimmunoprecipitation of in vitro
translated, radiolabeled Snp1p with Prp8p or Exo84p.
FLAG-tagged Snp1p, Exo84p, and a 115-kDa fragment of HA-tagged Prp8
(Prp8f) were synthesized in a rabbit reticulocyte lysate in the
presence of [35S]methionine (Exo84p, lanes 2 and 14; Prp8f, lanes 4 and 6; and
Snp1p, lanes 5, 7, and 15). As controls, the
following were added to the translation reactions: no exogenous RNA,
lanes 1 and 8; and luciferase RNA, lane
3. The individual translations for HA-Prp8f, Exo84p, and
FLAG-Snp1p were then treated with RNase A after which equal volumes of
the reactions were mixed in the combinations indicated. The mixtures
were incubated at 30 °C for 30 min and then at 4 °C with
Sepharose beads with either anti-HA (lanes 11-13) or
anti-FLAG (lanes 16, 18, and 19) monoclonal
antibody (mAb) covalently attached or without antibody
(lanes 9, 10, and 17). Any immunoprecipitated
proteins were eluted and fractionated by SDS-PAGE and visualized by
fluorography as shown here. One-tenth of each of the total translation
that was used for the coimmunoprecipitations was loaded in lanes
6, 7, 14, and 15.
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Fig. 3.
The exo84-2 mutation confers
temperature-sensitive growth. The isogenic, haploid, wild type,
and exo84-2 mutant strains were grown overnight in YPD at
30 °C, resuspended in water, and streaked onto YPD medium, after
which the cultures were incubated at the indicated temperatures. Growth
was recorded after 3 days.
View larger version (45K):
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Fig. 4.
Primer extension analyses of RNAs transcribed
from chromosomal genes. Wild type and exo84-2 mutant
strains were grown to mid-log phase at the permissive temperature
(26 °C). The cultures were shifted to the non-permissive temperature
(37 °C), and samples were removed at the indicated times after the
shift. As a control, the Ts prp5-3 mutant strain was grown
at either 23 or 26 °C, and samples were removed at either 4 or 2 and
4 h, respectively after shift to 37 °C. The RNAs extracted from
the samples were analyzed by primer extension using probes specific for
ScR1, the control RNA, and the ribosomal protein Rpl30 (A),
actin (B), and the snRNA U5 (C). The reaction
products were separated by denaturing PAGE and visualized by
autoradiography as shown here. Radiolabeled MspI restriction
endonuclease fragments of pBR322 DNA were used as size markers
(M). D, the levels of mature RNAs were measured
by primer extension analyses in samples such as those in
A-C. The levels of mature Rpl30, actin, and U5 RNAs were
normalized to the levels of ScR1 RNA in each primer extension reaction.
The normalized values are expressed here as the percentage of
normalized RNA present at 0 h for either the mutant or wild type
strain. The means (±S.D.) from three exo84-2 mutant
isolates and three (for actin and ScR1) or two (for Rpl30) wild type
isolates are shown here. Two each of exo84-2 mutant and wild
type isolates were analyzed for U5 RNA levels. RNA levels in the mutant
SRYprp5-3a strain were measured twice. The probabilities calculated by
ANOVA that the means are equal in the wild type and mutant strains at
37 °C are indicated. The non-parametric Mood's Median test for the
Rpl30 levels at 4 h at 37 °C gave similar results
(p < 0.03).
View larger version (13K):
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Fig. 5.
Levels of Rpl30 and actin pre-mRNAs in
wild type and exo84-2 mutant strains at 26 and
37 °C. The levels of pre-mRNAs and mRNAs in cells
removed immediately before or at 1-4 h during incubation at 37 °C
were assayed by primer extension (Fig. 4, A and
B) and measured with a Molecular Dynamics PhosphorImager.
Three each of wild type and exo84-2 mutant yeast isolates
were assayed. The percent pre-mRNA equals (100 × units
pre-mRNA/(units pre-mRNA + mRNA)). The probabilities of the
means in the mutant and wild type strains at 37 °C being equal are
indicated by asterisks.
View larger version (67K):
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Fig. 6.
The exo84-2 mutant splicing
extract is heat-sensitive in vitro and complements
other inactivated mutant extracts. A, the isogenic
exo84-2 mutant and wild type WCE were incubated at 41 °C
for the indicated times (min) after which they were assayed for
splicing activity at 23 °C for 10 min with radiolabeled actin
pre-mRNA as substrate. The radiolabeled RNAs in the reactions were
extracted, fractionated by denaturing PAGE, and visualized by
autoradiography as shown here. The symbols from
top to bottom represent splicing intermediate
lariat-exon 2, lariat product, pre-mRNA, spliced mRNA, and
intermediate exon 1. B, active WCE from mutants
exo84-2, prp2-1, prp5-3, and
prp9-1 (84, 2, 5, and 9; lanes
1-4) were heat-inactivated in vitro (lanes
5-8). The inactivated extracts were then mixed pairwise in
various combinations (lanes 9-14). Splicing assays were
initiated by the addition of splicing buffer components, ATP, and
radiolabeled pre-mRNA and incubated for 15 min at 23 °C. The
RNAs were analyzed as in A.
View larger version (43K):
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Fig. 7.
The splicing defect caused by the
exo84-2 mutation can be complemented in
vitro with a fraction enriched for wild type Exo84p.
A, Western blot analysis of HA-tagged Exo84p in WCE and
extract fractions 40P3 and 40W. The fractions 40P3 (P3) and
40W (W), and WCE (CE) were made from a yeast
strain with HA-tagged Exo84p (+HA tag). Aliquots of these
extracts as well as WCE from a wild type strain ( HA tag)
were fractionated by SDS-PAGE, electrophoretically transferred to a
membrane, and probed with anti-HA monoclonal and anti-Bcy1 polyclonal
antibodies. Ha-tagged Exo84p and the control, Bcy1p (105) which is not
a spliceosomal factor, were visualized by chemiluminescence as shown
here. The following amounts of extracts were loaded onto the gel: 47 µg of CE, lane 1; 14 µg of P3, lane 2; 28 µg of P3, lane 3; 64 µg of P3, lane 4; 22 µg of W, lane 5; and 40 µg of CE (HA tag),
lane 6. B, in vitro complementation of
heat-inactivated exo84-2 mutant WCE with a fraction enriched
for wild type Exo84p. Active exo84-2 WCE (lane 1)
was heat-inactivated for 7 min at 41 °C. The inactivated WCE was
then combined with buffer D only (lane 2), wild type 40P3
(0.02 µg, lane 3; 1.7 µg, lane 4; 4.2 µg,
lane 5; and 16.7 µg, lane 6) or wild type 40W
(5 µg, lane 7; 25 µg, lane 8; and 50 µg,
lane 9) and assayed for splicing activity using radiolabeled
actin pre-mRNA as described in Fig. 6A. As controls, the
active mutant extract was assayed with added 40P3 (16.7 µg;
lane 10) or 40W (50 µg; lane 11). The fractions
40P3 (16.7 µg, lane 12) and 40W (100 µg, lane
13) individually had no splicing activity.
,
, and , with the and bands migrating closely to one
another. We found that the untreated mutant and both the untreated and
heat-treated wild type extracts formed all five spliceosomal complexes
that contribute to these three bands (Fig.
8). In contrast, the inactivated mutant extract formed the ATP-independent complex but little or none of
the other complexes. Thus the exo84-2 mutation blocks
prespliceosome formation.
View larger version (96K):
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Fig. 8.
The inactivated exo84-2
mutant extract is defective in prespliceosome formation.
Active and heat-treated exo84-2 and wild type WCE were
assayed for spliceosome assembly activity as shown here. Half of each
wild type and mutant extract was heated at 41 °C for 7 min. Splicing
buffer components, ATP, and radiolabeled actin pre-mRNA were then
added to the untreated and treated extracts and incubated at 23 °C.
At the times indicated, samples were removed, quenched on ice, and then
separated by native PAGE. Splicing-specific complexes containing
radiolabeled RNA were visualized by autoradiography as shown here. The
sequence of complex formation on the pre-mRNA is 
1 (the prespliceosome) 2 1 2 (the active spliceosome) (9);
however, the individual complexes within the and bands are not
distinguishable in assays using radiolabeled pre-mRNA as substrate.
The complex contains U1 snRNP. The 1 complex (the
prespliceosome) contains both U1 and U2 snRNPs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or commitment complex (3, 5). Furthermore,
factors bound to the branchpoint region of pre-mRNA (83), as well
as the activities of Prp5p (45, 84) and at least seven other proteins
including Prp9p, are important for U2 snRNP binding and prespliceosome
formation (4, 85). Therefore, inactivation of Exo84p may affect the complex, the U2 snRNP, or some other factor in the extract that
prevents the U2 snRNP from stably associating with the complex.
However, we were unable to detect a physical association of Exo84p with
any of the spliceosomal snRNPs. The interactions of Exo84p with these
factors may be transient or unstable and therefore hard to detect. We
must also consider that Exo84p could modify or be a cofactor for
modifying a spliceosomal factor.
ACKNOWLEDGEMENTS
FOOTNOTES
Current address: Lovelace Respiratory Research Institute,
Albuquerque, NM 87185-5890.
ABBREVIATIONS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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